The present invention relates to the field of immunology and, in particular, to two new products; chimeric and humanized monoclonal antibodies against epidermal growth factor receptor (xe2x80x9cEGF-Rxe2x80x9d). The chimeric and humanized monoclonal antibodies of the present invention are less immunogenic than original murine monoclonal antibodies and possess improved effector functions.
The present invention also relates to therapeutic and diagnostic compositions comprising these antibodies.
The Epidermal Growth Factor (xe2x80x9cEGFxe2x80x9d) is a 53 amino acid polypeptide with a molecular weight of 6045 D and was isolated and purified, for the first time, from murine submaxillary gland. (Cohen S; J Biol Chem (1962) 237, 1555). Later, a similar molecule was obtained from human urine. (Cohen S and Carpenter G; (1975) PNAS USA 72, 1317). The activity of EGF is primarily performed via its interaction with its membrane receptor, a 170 kDa molecular weight glycoprotein. The receptor""s intracellular domain is associated with a tyrosine kinase activity and has structural homology to the oncogene v-erb-B showing relation to the malignant transformation process (Heldin C H et al; (1984) Cell 37, 9-20).
High levels of EGF-R have been detected in malignant tumors of epithelium origin such as breast, bladder, ovarian, vulva, colonic, lung, brain and esophagus cancers. The role of EGF and its receptor in regulating tumor growth is unknown, but it has been suggested that EGF-R expression in tumor cells provides a mechanism for autocrine growth stimulation which leads to uncontrolled proliferation (Schlessinger J, Schreiber A B, Levi A, Liberman T and Yarden Y; (1983) Crit Rev Biochem 14(2), 93-111).
It has been reported that EGF produces overgrowth of breast cancer cell lines (Osborne C K et al; (1980) Cancer Research 40, 236 1), as well as modulating the differentiation under some cellular systems (Tonelli C J; Nature (1980) 285, 250-252). These effects on cellular differentiation and proliferation are related to the high expression of EGF-R (Buss J E et al; (1982) PNAS 79, 2574).
The presence of EGF-R in tumor cells has proven to be an indicator of a poor prognosis in human breast cancer. Approximately 40% of the breast tumors show specific binding sites having high affinity for EGF suggesting this growth factor receptor could broaden the concept of hormone dependency in breast cancer (Perez R, Pascual M R, Macias A, Lage A; (1984) Breast Cancer Research and Treatment 4, 189-193). There is also an inverse correlation between the expression of EGF-R and the presence of estrogen receptor, indicating EGF-R as an indifferentiation marker or an indicator of the potential for proliferation of the malignant cells.
Other groups have reported that the expression of EGF-R is higher in regional ganglionar metastasis than in primary carcinomas of breast (Sainsbury J R et al; (1985) Lancet 1, 8425, 364-366) and that the expression of the receptor is different in the different histologic subtypes of human breast carcinoma cells, where presence of the receptor constitutes a signal of a poor prognosis (Macias A et al; Anti Cancer Research 6: 849-852).
The results obtained in different studies have prompted the consideration of the EGF/EGF-R system as a possible target for therapeutic actions.
We obtained a murine monoclonal antibody (R3), raised against the human placenta as described in European Patent application No. 93202428.4, and found to bind to the external domain of the human EGF-R. The R3 antibody was found to inhibit the binding of EGF at both low and high affinity EGF-R sites.
Passive immunotherapy using monoclonal antibodies against the EGF-R have been the object of multiple investigations and have demonstrated that the specific recognition of the receptor by the antibody inhibits the EGF binding and has an inhibitory effect on the mitogenic stimulation of malignant cells (Sato J D et al; (1987) Methods in Enzimology 148, 63-81). However, there is evidence that the murine origin of these antibodies produces a human anti-mouse antibody response.
The development of the hybridoma antibody technique by Kohler and Milstein revolutionized the discipline of immunochemistry and provided a new family of reagents with potential applications in clinical diagnosis and immunotherapy (Kohler G, Milstein C; (1975) Nature 256, 495-497). While it has become routine to produce mouse monoclonal antibodies (mAbs) for use in basic research and clinical diagnosis, it has been difficult to use these mAbs for in vivo immunotherapy because they have reduced half-life in humans, there is poor recognition of mouse antibody effector domains by the human immune system, and the foreign immunoglobulin can elicit an antiglobulin response (HAMA response) that may interfere with therapy.
The ability to genetically manipulate antibody genes and then express these altered genes by transfection techniques enables us to produce mAbs having more desirable properties than the existing hybridoma antibodies. Thus, genetic engineering can be used to enhance desired effector functions in antibody molecules and to decrease or eliminate undesired effector functions.
The use of recombinant DNA technology to clone antibody genes has provided an alternative wherein a murine mAb can be converted to a predominantly human form with the same antigen binding properties. In 1984, S L Morrison created mouse-human antibody molecules, of defined antigen-binding specificity, by taking the variable regions genes of mouse antibody producing myeloma cell lines, and joining them to human immunoglobulin constant regions (Morrison S L et al; (1984) PNAS USA 81, 6851-6855).
Other authors have attempted to build rodent antigen binding sites directly into human antibodies by transplanting only the antigen binding site, rather than the entire variable domain, from a murine antibody (Jones P T et al; (1986) Nature 321, 522-524; Verhoeven M et al; (1988) Science 239, 1534-1536). Some applications of this method have been developed (Rietchmann L et al; (1988) Nature 332, 323-327; Quee C et al; (1989) PNAS USA 86, 10029-10033), other authors have worked with reshaped antibodies, which included some murine residues in human FRs in order to recover the affinity for the original antigen (Tempest PR; (1991) Biotechnology 9, 266-272).
Orlandi R et al. (Proc Natl Acad Sci USA 86, 3833-3837, 1989) disclose the constant regions of the human gamma-1 heavy chain and the human kappa light chain, and suitable cloning vectors thereof.
The invention provides a chimeric and a humanized mAb which are directed to the EGF-R comprising, an antigen-binding site of non-human origin, and the constant regions of human origins (chimeric) and the framework regions (xe2x80x9cFRsxe2x80x9d) of the variable regions and the constant regions of human origins may be, if necessary, modified in a way that the specificity of the binding to EGR-R can be conserved or restored.
The present invention can be used to characterize the hypervariable regions of the antigen-binding site of an antibody against the EGF-R and provide these CDRs within a humanized and chimeric mAb defined as above.
These antibodies can play a role as a therapeutic or diagnostic agent in order to combat tumors with high expression of EGF-R.
The present invention also provides chimeric and humanized antibodies specific for the EGF-R.
More specifically, the invention provides a chimeric monoclonal antibody comprising variable regions of non-human origin and constant regions of light and heavy chains of human origin, wherein the chimeric monoclonal antibody binds to human EGF-R and inhibits binding of EGF to EGF-R. According to a preferred embodiment of the chimeric monoclonal antibody, the variable regions of the antigen binding sites comprise the amino acid sequences shown in FIGS. 1a and 1b. 
Further, the invention provides a humanized monoclonal antibody comprising antigen binding sites (CDRs) of non-human origin and the FRs of variable region and constant regions of light and heavy chains of human origin, wherein the humanized monoclonal antibody binds to human EGF-R and inhibits binding of EGF to EGF-R. According to a preferred embodiment of the humanized monoclonal antibody, the hypervariable regions of the antigen binding sites comprise the amino acid sequences underlined in FIG. 1 and FIG. 1b. Preferably, the FRs of the variable region which is not related to the antigen binding sites comprise the following amino acid sequences:
light chain:
FR1: asp-ie-gln-met-thr-gln-ser-pro-ser-ser-leu-ser-ala-ser-val-gly-asp-arg-val-thr-ile-thr-cys (SEQ ID NO: 1),
FR2: trp-tyr-gln-gln-thr-pro-gly-lys-ala-pro-lys-leu-leu-ile-tyr (SEQ ID NO: 2),
FR3: gly-val-pro-ser-arg-phe-ser-gly-ser-gly-ser-gly-thr-asp-phe-thr-phe-thr-ile-ser-ser-leu-gln-pro-glu-asp-ile-ala-thr-tyr-tyr-cys (SEQ ID NO: 3),
FR4: phe-gly-gln-gly-thr-lys-leu-gln-ile-thr-arg-glu (SEQ ID NO: 4),
FR1: gln-val-gln-leu-gln-gln-ser-gly-ala-glu-val-lys-lys-pro-gly-ser-ser-val-lys-val-ser-cys-lys-ala-ser-gly-tyr-thr-phe-thr (SEQ ID NO: 5),
FR2: trp-val-arg-gln-ala-pro-gly-gln-gly-leu-glu-trp-ile-gly (SEQ ID NO: 6),
FR3: (arg,lys)-(val,ala)-thr-ile-thr-val-asp-glu-ser-(thr,ser)-(thr,asn)-thr-ala-tyr-met-glu-leu-ser-ser-leu-arg-ser-glu-asp-thr-ala-phe-tyr-phe-cys-(ala,thr)-arg (SEQ ID NO: 7),
FR4: trp-gly-gln-gly-ser-thr-val-thr-val-ser-ser (SEQ ID NO: 8), and wherein the amino acids listed in brackets are alternatives.
The humanized monoclonal antibody may comprise a derivative of an amino acid sequence modified by amino acid substitution within the variable and constant regions wherein the biological function of specific binding to the antigen is preserved.
In a preferred embodiment of the humanized and chimeric monoclonal antibodies of the present invention, the constant region of the heavy chain comprises the amino acid sequence of a gamma-1 chain and the constant regions of the light chain comprise the amino acid sequences of a kappa chain of a human immunoglobulin. A purified humanized or chimeric monoclonal antibody which derives from murine mAb R3 is preferred.
The invention also includes a pharmaceutical composition comprising a chimeric or humanized monoclonal antibody as defined herein. Usually, the composition will also contain a pharmaceutically acceptable carrier.
The invention also includes in the use of a humanized or chimeric antibody, as defined herein, for the manufacture of a drug directed to tumors, and use of a humanized or chimeric antibody, as defined herein, for diagnostic localization and assessing tumor growth.